A complex gene cluster for indole-diterpene biosynthesis in the grass endophyte Neotyphodium lolii

Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloë/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.     

Effects of the fungal endophyte, Neotyphodium lolii, on net photosynthesis and growth rates of perennial ryegrass (Lolium perenne) are independent of In Planta endophyte concentration

• Background and Aims Neotyphodium lolii is a fungal endophyteof perennial ryegrass (Lolium perenne), improving grass fitnessthrough production of bioactive alkaloids. Neotyphodium speciescan also affect growth and physiology of their host grasses(family Poaceae, sub-family Pooideae), but little is known aboutthe mechanisms. This study examined the effect of N. lolii onnet photosynthesis (P_n) and growth rates in ryegrass genotypesdiffering in endophyte concentration in all leaf tissues. • Methods Plants from two ryegrass genotypes, Nui D andNui UIV, infected with N. lolii (E+) differing approx. 2-foldin endophyte concentration or uninfected clones thereof (E–)were grown in a controlled environment. For each genotype xendophyte treatment, plant growth rates were assessed as tilleringand leaf extension rates, and the light response of P_n, darkrespiration and transpiration measured in leaves of young (30–45d old) and old (>90 d old) plants with a single-chamber openinfrared gas-exchange system. • Key Results Neotyphodium lolii affected CO₂-limited ratesof P_n, which were approx. 17 % lower in E+ than E– plants(P < 0·05) in the young plants. Apparent photon yieldand dark respiration were unaffected by the endophyte (P >0·05). Neotyphodium lolii also decreased transpiration(P < 0·05), but only in complete darkness. There wereno endophyte effects on P_n in the old plants (P > 0·05).E+ plants grew faster immediately after replanting (P < 0·05),but had approx. 10 % lower growth rates during mid-log growth(P < 0·05) than E– plants, but there was noeffect on final plant biomass (P > 0·05). The endophyteeffects on P_n and growth tended to be more pronounced in NuiUIV, despite having a lower endophyte concentration than NuiD. • Conclusions Neotyphodium lolii affects CO₂ fixation,but not light interception and photochemistry of P_n. The impactof N. lolii on plant growth and photosynthesis is independentof endophyte concentration in the plant, suggesting that theendophyte mycelium is not simply an energy drain to the plant.However, the endophyte effects on P_n and plant growth are stronglydependent on the plant growth phase.     

Neotyphodium endophyte strain and superoxide dismutase activity in perennial ryegrass plants under water deficit

The presence of Neotyphodium endophyte in forage grass tillers has been associated with increased tolerance of abiotic stresses. The effect of four endophyte treatments (plants with three different strains of Neotyphodium lolii compared with plants without endophyte) on superoxide dismutase (SOD) (EC1.15.1.1) activity in Lolium perenne cv ‘Grasslands Samson’ was measured under high and low dehydration regimes in a glasshouse experiment. SOD activity was assayed by a microplate method utilising the inhibition of reduction of a tetrazolium dye by superoxide radicals. A progressive increase in dehydration over 2 weeks reduced shoot fresh weight, dry weight and shoot water content for high, compared with low, moisture-stressed plants. Mean shoot fresh weight was significantly lower for plants with strain AR37 endophyte than for plants with strain AR1 endophyte, wild-type or endophyte-free plants, but there was no interaction between endophyte treatment and dehydration treatment. There were no differences in mean SOD activity between the dehydration treatments, and the four endophyte treatments at any of the harvests. All harvest mean SOD levels for plants in both stress groups, however, were significantly different from the preceding harvest value. Between the first and second week of moisture stress there was a significant endophyte by harvest interaction for mean percentage change in SOD activity when activity in plants with wild-type strain endophyte increased more rapidly than in AR1, AR37 or endophyte-free plants. The results are in agreement with earlier reports suggesting that Neotyphodium endophytes do not have major effects on the water stress physiology of perennial ryegrass, although water deficits applied were not extreme.     

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.     

Advanced data-mining strategies for the analysis of direct-infusion ion trap mass spectrometry data from the association of perennial ryegrass with its endophytic fungus, Neotyphodium lolii

Direct-infusion mass spectrometry (MS) was applied to study the metabolic effects of the symbiosis between the endophytic fungus Neotyphodium lolii and its host perennial ryegrass (Lolium perenne) in three different tissues (immature leaf, blade, and sheath). Unbiased direct-infusion MS using a linear ion trap mass spectrometer allowed metabolic effects to be determined free of any preconceptions and in a high-throughput fashion. Not only the full MS(1) mass spectra (range 150-1,000 mass-to-charge ratio) were obtained but also MS(2) and MS(3) product ion spectra were collected on the most intense MS(1) ions as described previously (Koulman et al., 2007b). We developed a novel computational methodology to take advantage of the MS(2) product ion spectra collected. Several heterogeneous MS(1) bins (different MS(2) spectra from the same nominal MS(1)) were identified with this method. Exploratory data analysis approaches were also developed to investigate how the metabolome differs in perennial ryegrass infected with N. lolii in comparison to uninfected perennial ryegrass. As well as some known fungal metabolites like peramine and mannitol, several novel metabolites involved in the symbiosis, including putative cyclic oligopeptides, were identified. Correlation network analysis revealed a group of structurally related oligosaccharides, which differed significantly in concentration in perennial ryegrass sheaths due to endophyte infection. This study demonstrates the potential of the combination of unbiased metabolite profiling using ion trap MS and advanced data-mining strategies for discovering unexpected perturbations of the metabolome, and generating new scientific questions for more detailed investigations in the future.     

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.     

Effects of biotic and abiotic stress on the growth of three genotypes of Lolium perenne with and without infection by the fungal endophyte Neotyphodium lolii

The combinations of three genotypes of Lolium perenne with and without (i) infection by the fungal endophyte Neotyphodium lolii, (ii) infection by ryegrass mosaic virus and (iii) one of five different forms of abiotic stress were studied in pot experiments in a glasshouse. The five abiotic stress treatments were (i) low pH (compared with ‘optimal’ pH), (ii) cutting plants to a height of 1 cm (compared with 5 cm), (iii) shading (compared with no shading), (iv) cutting plants at 2‐weekly intervals (compared with 6 wk) and (v) low nitrogen applied (compared with ‘high’ nitrogen applied). On average, over the five experiments, the accumulated herbage dry weight was 10% more for N. lolii‐infected plants than uninfected, 22% more for virus‐free plants than infected, and 265% more for ‘unstressed’ plants than for plants with abiotic stress. The effects of N. lolii infection on plant growth when the plants were under abiotic or biotic stress were not consistent.     

Confining mycelial growth to porous microbeads: a novel technique to alter the morphology of non-newtonian mycelial cultures

In an effort to alter the filamentous morphology of Penicillium chrysogenum cells, a technique was developed to confine the growth of the mycelia to porous celite beads. The pore matrix of these beads was found to be very effective for entrapping mycelial cells and spores. The entrapped spores were used to initiate the fermentations in shake flask cultures. Significant increases in final cell densities were obtained in the confined cell cultures reaching up to 60 g/L cells. This is nearly double the cell concentration attainable in free cell cultures grown in the absence of beads. Cell loadings up to 0.55 g cells per bead were obtained in the confined cell cultures. In the later stages of the fermentations, the specific oxygen uptake rates in the confined cell cultures were found to decrease with respect to free cell cultures.     

Resistance to the rice leaf bug, Trigonotylus caelestialium, is conferred by Neotyphodium endophyte infection of perennial ryegrass, Lolium perenne

Neotyphodium fungal endophytes form mutualistic symbiotic associations with many grasses of the subfamily Pooideae, including important forage and turfgrass species. This relationship provides a competitive advantage to the host plant by increasing abiotic/biotic stress tolerance, such as its resistance to drought, diseases, and insect pests. The insect deterrent effects of endophytes are now receiving attention in Japan, as insect pests growing in meadows are causing problems in adjacent rice paddies. One of the most serious problems is the kernel spotting of rice grains caused by the rice leaf bug, Trigonotylus caelestialium Kirkaldy (Heteroptera: Miridae), which reproduces on Lolium species grown as forage. To determine the potential of Neotyphodium endophytes to reduce the invasion of rice crops by T. caelestialium from adjacent Lolium crops, we carried out choice and no‐choice feeding tests using endophyte‐infected and endophyte‐free clonal perennial ryegrass (Lolium perenne L.) (Poaceae). Our experiments revealed that the presence of the Neotyphodium endophyte strongly deterred the feeding of both first‐instar larvae and adults of T. caelestialium. These results show the potential of Neotyphodium endophytes to reduce the number of T. caelestialium in forage fields and grasslands, and thus to reduce the damage to rice grains caused by this insect pest.     

Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii.

Lysergyl peptide synthetase 1 catalyzes the assembly of toxic ergopeptines from activated D-lysergic acid and three amino acids. The gene encoding this enzyme in the endophytic fungus Neotyphodium lolii was analyzed and compared to a homologous gene from the ergot fungus Claviceps purpurea. Each gene contained two introns, which were found in the same relative position within two modules of the gene. The 5' ends of the two genes were unusually divergent. Signature sequences determining substrate specificity were similar in adenylation domains that recognized identical amino acids but differed within the adenylation domain for the amino acid that varies between the major ergopeptines of the two fungi. Homologues were detected in several related endophytic fungi; the tall fescue endophyte Neotyphodium coenophialum contained a divergent, second copy of the gene. Our results provide new information on the structure and distribution of this important peptide synthetase involved in ergot alkaloid biosynthesis.     

Dehydration of germinating perennial ryegrass seeds can alter rate of subsequent radicle emergence

Perennial ryegrass (Lolium perenne L.) seeds (caryopses) germinateat or near the soil surface, where water potential can fluctuatewidely. This study examined germination of ‘Del Ray’perennial ryegrass seeds when imbibition was interrupted bydehydration prior to radicle emergence. Seeds were hydratedfor 0 to 40 h (26C), dehydrated at atmospheric water potentialsof –4, –40, –100 and/or –150 MPa for4–168 h, then rehydrated. Germination (radicle elongation 1 mm), seedling growth, solute leakage, and endogenous abscisicacid (ABA) levels were measured. Treatment differences in finalgermination percentage, seedling growth, and solute leakagewere generally not significant. However, the onset of radicleemergence was delayed and the rate of germination slowed whendehydration at –150 MPa was initiated after 36 or 40 hhydration. Slowed germination rates were not observed when dehydrationwas initiated before 36 h, when dehydration occurred at –4MPa, or when dehydration at –150 MPa was preceded by dehydrationat –4 MPa for 24 h. Exogenous abscisic acid (ABA) concentrationsabove 10^–6 M inhibited germination. However, endogenouswhole seed ABA levels declined during imbibition due to leaching,and did not increase during dehydration treatments that delayedgermination. These results illustrate that rate of late-occurringdehydration treatments is critical in determining subsequentgermination response. We propose that seed response to late-occurringdehydration may be of ecological significance in timing radicleemergence to coincide with adequate soil moisture for seedlingestablishment. Key words: Abscisic acid, seed germination, timing     

Gene flow in the endophyte Neotyphodium and implications for coevolution with Festuca arizonica

Arizona fescue (Festuca arizonica) often harbours asymptomatic, asexual endophytic fungi from the genus Neotyphodium. In agronomic grasses, Neotyphodium endophytes are often credited with a wide range of mutualistic benefits to its host many of which are related to fungal production of alkaloids for herbivore deterrence. Neotyphodium in the native grass Arizona fescue, however, usually produces alkaloids at levels too low to deter herbivores, and in general, does not behave mutualistically. This study uses microsatellite markers to examine rates of gene flow among four Arizona populations of Neotyphodium. Haplotypic diversity was generally low; only one population contained more than two haplotypes. Haplotypes carrying multiple loci for some or all of the microsatellite loci were also found, indicating a vegetative hybridization event between Neotyphodium and the grass choke pathogen from the genus Epichloë. Gene flow between Neotyphodium populations is very low, and likely much lower than the pollen mediated gene flow of its host. These differing rates of gene flow are predicted to create trait mismatching between endophyte and host and may explain the low, or lack of, alkaloid production by Neotyphodium in Arizona fescue and other native grass species.     

Effects of the Neotyphodium endophyte fungus on dormancy and germination rate of Lolium multiflorum seeds

Abstract Neotyphodium frequently occurs as an endophyte in grasses. Evidence shows enhanced fitness of endophyte infected grasses relative to non‐infected ones. Some studies of seed germination show endophyte enhancement of plant fitness in various environments, but inconsistent results indicate that further studies are needed. So far, experiments have failed to separate the confounded effects of population origin and seed management. For this reason, we evaluated the effects of endophyte infection on seed dormancy and germination in Lolium multiflorum using an experimental design controlling these factors. Depending on the year of seed production, endophyte infection modified seed response to light quality, affecting predominantly seed dormancy levels. Nevertheless, the endophyte did not affect base temperature or thermal time of germination. We concluded that endophytes were not a strong influence on germination behaviour. We speculate from our results that the presence of the endophyte changes germination by an indirect effect, in extending growth of the maternal plant during seed development and ripening. The direct effect of hyphae in the seed on seed behaviour was disregarded, because the difference between infected and non‐infected seed varied within the year of seed production. Future experiments should focus on effects of the endophyte on the canopy of parent plants during seed production and ripening, and, hence, on subsequent dormancy and germination of the seeds.     

Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epichlo? (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC-MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15-30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes.     

In planta reduction of maize seedling stalk lesions by the bacterial endophyte Bacillus mojavensis

Maize (Zea mays L.) is susceptible to infection by Fusarium verticillioides through autoinfection and alloinfection, resulting in diseases and contamination of maize kernels with the fumonisin mycotoxins. Attempts at controlling this fungus are currently being done with biocontrol agents such as bacteria, and this includes bacterial endophytes, such as Bacillus mojavensis . In addition to producing fumonisins, which are phytotoxic and mycotoxic, F. verticillioides also produces fusaric acid, which acts both as a phytotoxin and as an antibiotic. The question now is Can B. mojavensis reduce lesion development in maize during the alloinfection process, simulated by internode injection of the fungus? Mutant strains of B. mojavensis that tolerate fusaric acid were used in a growth room study to determine the development of stalk lesions, indicative of maize seedling blight, by co-inoculations with a wild-type strain of F. verticillioides and with non-fusaric acid producing mutants of F. verticillioides. Lesions were measured on 14-day-old maize stalks consisting of treatment groups inoculated with and without mutants and wild-type strains of bacteria and fungi. The results indicate that the fusaric-acid-tolerant B. mojavensis mutant reduced stalk lesions, suggesting an in planta role for this substance as an antibiotic. Further, lesion development occurred in maize infected with F. verticillioides mutants that do not produce fusaric acid, indicating a role for other phytotoxins, such as the fumonisins. Thus, additional pathological components should be examined before strains of B. mojavensis can be identified as being effective as a biocontrol agent, particularly for the control of seedling disease of maize.     

The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere

Members of the genus Neotyphodium are asexual, seedborne, protective fungal endophytes of cool season grasses that have likely evolved either directly from sexual Epichlo?; species, or by the interspecific hybridization of distinct lineages of Epichlo?; and Neotyphodium. We investigated the evolutionary origins of Neotyphodium endophytes from several grasses that are indigenous to the Southern Hemisphere using a multiple-gene phylogenetic approach. Intron regions of the genes encoding β-tubulin (tub2), translation elongation factor 1-α (tef1) and actin (act1) were amplified by polymerase chain reaction and sequenced. Phylogenetic analyses of these sequences, aligned with homologous sequences from Epichlo?; spp., revealed the evolutionary origins of the Southern Hemisphere endophytes, where one lineage of apparently non-hybrid origin, and three lineages of unique interspecific hybrid origin were identified. On the basis of morphology, host range and evolutionary history, we propose three new species of Neotyphodium. Neotyphodium aotearoae was isolated from Echinopogon ovatus populations from New Zealand and Australia, and comprised a unique, apparently non-hybrid lineage within the Epichlo?; species phylogeny. In contrast, an interspecific hybrid lineage was identified from two Australian Ec. ovatus populations, whose ancestry apparently involved lineages closely related to extant E. festucae and an E. typhina genotype similar to that of isolates from Poa pratensis. Endophytes infecting South African Melica racemosa and M. decumbens (dronkgras) appeared to be hybrids of E. festucae and N. aotearoae or close relatives. The names N. australiense and N. melicicola are proposed for these two hybrid lineages, respectively. The origin of N. tembladerae, an established endophyte species from South American Poa and Festuca spp., was also investigated. Neotyphodium tembladerae appeared to be of hybrid origin, involving E. festucae and an E. typhina genotype similar to that of isolates from Poa nemoralis. The results of this study highlight the widespread occurrence of interspecific hybrid Neotyphodium lineages on a global scale, and the extent of endophyte gene-flow between the Northern and Southern Hemispheres.     

Dynamics of Neotyphodium endophyte infection in ageing seed pools: incidence of differential viability loss of endophyte,infected seed and non-infected seed

Symbiotic associations between grasses and vertically transmitted endophytic fungi are widespread in nature. Within grass populations, changes in the frequency of infected plants are driven by influence of the endophyte on the fitness of their hosts and by the efficiency of endophyte transmission from parent plants to their offspring. During the seed stage, the endophyte might influence the fitness of its host by affecting the rate of seed viability loss, whereas the efficiency of endophyte transmission is affected by losses of viability of the fungus within viable seeds. We assessed the viability losses of Lolium multiflorum seeds with high and low level of infection of the endophyte Neotyphodium occultans, as well as the loss of viability of the fungus itself, under accelerated seed ageing and under field conditions. Starting with high endophyte-infected accessions of L. multiflorum, we produced their low endophyte-infected counterparts by treating seeds with a fungicide, and subsequently multiplying seeds in adjacent plots allowing pollen exchange. In our accelerated ageing experiments, which included three accessions, high endophyte-infected seeds lost viability significantly faster than their low endophyte-infected counterpart, for only one accession. High endophyte-infected seeds of this particular accession absorbed more water than low endophyte-infected seeds. In contrast, the endophyte lost viability within live seeds of all three accessions, as the proportions of viable seeds producing infected seedlings decreased over time. In our field experiment, which included only one accession, high endophyte-infected seed lost viability significantly but only slightly faster than low endophyte-infected seed. In contrast, the loss of viability of the endophyte was substantial as the proportions of viable seeds producing infected seedlings decreased greatly over time. Moving the seeds from the air to the soil surface (simulating seed dispersion off the spikes) decreased substantially the rate of seed viability loss, but increased the rate of endophyte viability loss. Our experiments suggest that, in ageing seed pools, endophyte viability loss and differential seed mortality determine decreases in the proportions of endophyte-infected seeds in L. multiflorum. Endophyte viability loss within live seeds contributes substantially more to infection frequency changes than differential viability losses of infected and non-infected seeds.     

Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter

The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape.     

Enhanced resistance to four species of Clypeorrhynchan pests in Neotyphodium uncinatum infected Italian ryegrass

Particular alkaloids produced by Neotyphodium endophytes show toxicity to invertebrates. Italian ryegrass (Lolium multiflorum Lamarck) cultivars and strains that are symbiotic with Neotyphodium endophytes have been recently established in Japan. N. uncinatum-infected Italian ryegrass lines accumulate N-formylloline, a type of loline alkaloid (1-aminopyrrolizidine) showing neurotoxicity to herbivorous insects. This study investigated the toxicity of N-formylloline and resistance of N. uncinatum-infected Italian ryegrass to vascular-sap feeding Clypeorrhynchan pests. When four vascular-sap feeding insects: Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), Sogatella furcifera (Horváth) (Homoptera: Delphacidae), Cicadulina bipunctata (Melichar) (Homoptera: Cicadellidae), and Nephotettix cincticeps (Uhler) (Homoptera: Cicadellidae) fed on N. uncinatum-infected Italian ryegrass, significant decreases in survival rate were observed for three phloem-sap feeders but not for a xylem-sap feeder, N. cincticeps. This result suggests an uneven distribution of N-formylloline among plant tissues. A potency assay for N-formylloline using a Parafilm feeding sachet and a quantitative analysis of N-formylloline in plant showed a concentration-dependent lethal effect of N-formylloline on all four tested vascular-sap feeders. Our results strongly suggest that N. uncinatum-infected plants can control some Clypeorrhynchan pests in crop fields.